The double inhibition of endogenously produced BMP and Wnt factors synergistically triggers dorsal telencephalic differentiation of mouse ES cells

Embryonic stem (ES) cells are becoming a popular model of in vitro neurogenesis, as they display intrinsic capability to generate neural progenitors that undergo the known steps of in vivo neural development. These include the acquisition of distinct regional fates, which depend on growth factors and signals that are present in the culture medium. The control of the intracellular signaling that is active at different steps of ES cell neuralization, even when cells are cultured in chemically defined medium, is complicated by the endogenous production of growth factors. However, this endogenous production has been poorly investigated so far. To address this point, we performed a high‐throughput analysis of the expression of morphogens during mouse ES cell neuralization in minimal medium. We found that during their neuralization, ES cells increased the expression of members of Wnt, Fibroblast Growth Factor (FGF), and BMP families. Conversely, the expression of Activin/Nodal and Shh ligands was low in early steps of neuralization. In this experimental condition, neural progenitors and neurons generated by ES cells expressed a gene expression profile that was consistent with a midbrain identity. We found that endogenous BMP and Wnt signaling, but not FGF signaling, synergistically affected ES cell neural patterning, by turning off a profile of dorsal/telencephalic gene expression. Double BMP and Wnt inhibition allowed neuralized ES cells to sequentially activate key genes of cortical differentiation. Our findings are consistent with a novel synergistic effect of Wnt and BMP endogenous signaling of ES cells in inhibiting a cortical differentiation program. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 66–79, 2015     

Coordinated regulation of the dorsal‐ventral and anterior‐posterior patterning of Xenopus embryos by the BTB/POZ zinc finger protein Zbtb14

During early vertebrate embryogenesis, bone morphogenetic proteins (BMPs) belonging to the transforming growth factor‐β (TGF‐β) family of growth factors play a central role in dorsal–ventral (DV) patterning of embryos, while other growth factors such as Wnt and fibroblast growth factor (FGF) family members regulate formation of the anterior–posterior (AP) axis. Although the establishment of body plan is thought to require coordinated formation of the DV and AP axes, the mechanistic details underlying this coordination are not well understood. Here, we show that a Xenopus homologue of zbtb14 plays an essential role in the regulation of both DV and AP patterning during early Xenopus development. We show that overexpression of Zbtb14 promotes neural induction and inhibits epidermal differentiation, thereby regulating DV patterning. In addition, Zbtb14 promotes the formation of posterior neural tissue and suppresses anterior neural development. Consistent with this, knock‐down experiments show that Zbtb14 is required for neural development, especially for the formation of posterior neural tissues. Mechanistically, Zbtb14 reduces the levels of phosphorylated Smad1/5/8 to suppress BMP signaling and induces an accumulation of β‐Catenin to promote Wnt signaling. Collectively, these results suggest that Zbtb14 plays a crucial role in the formation of DV and AP axes by regulating both the BMP and Wnt signaling pathways during early Xenopus embryogenesis.     

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.     

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17⁺ endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17⁺ cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP⁺ cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17⁺ cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.     

Frizzled7 mediates canonical Wnt signaling in neural crest induction

The neural crest is a multipotent cell population that migrates from the dorsal edge of the neural tube to various parts of the embryo where it differentiates into a remarkable variety of different cell types. Initial induction of neural crest is mediated by a combination of BMP, Wnt, FGF, Retinoic acid and Notch/Delta signaling. The two-signal model for neural crest induction suggests that BMP signaling induces the competence to become neural crest. The second signal involves Wnt acting through the canonical pathway and leads to expression of neural crest markers such as slug. Wnt signals from the neural plate, non-neural ectoderm and paraxial mesoderm have all been suggested to play a role in neural crest induction. We show that Xenopus frizzled7 (Xfz7) is expressed in the dorsal ectoderm including early neural crest progenitors and is a key mediator of the Wnt inductive signal. We demonstrate that Xfz7 expression is induced in response to a BMP antagonist, noggin, and that Xfz7 can induce neural crest specific genes in noggin-treated ectodermal explants (animal caps). Morpholino-mediated or dominant negative inhibition of Xfz7 inhibits Wnt induced Xslug expression in the animal cap assay and in the whole embryo leading to a loss of neural crest derived pigment cells. Full-length Xfz7 rescues the morpholino-induced phenotype, as does activated beta-catenin, suggesting that Xfz7 is signaling through the canonical pathway. We therefore demonstrate that Xfz7 is regulated by BMP antagonism and is required for neural crest induction by Wnt in the developing vertebrate embryo.     

Cell‐autonomous signal transduction in the Xenopus egg Wnt/β‐catenin pathway

Wnt proteins are thought to bind to their receptors on the cell surfaces of neighboring cells. Wnt8 likely substitutes for the dorsal determinants in Xenopus embryos to dorsalize early embryos via the Wnt/β‐catenin pathway. Here, we show that Wnt8 can dorsalize Xenopus embryos working cell autonomously. Wnt8 mRNA was injected into a cleavage‐stage blastomere, and the subcellular distribution of Wnt8 protein was analyzed. Wnt8 protein was predominantly found in the endoplasmic reticulum (ER) and resided at the periphery of the cells; however, this protein was restricted to the mRNA‐injected cellular region as shown by lineage tracing. A mutant Wnt8 that contained an ER retention signal (Wnt8‐KDEL) could dorsalize Xenopus embryos. Finally, Wnt8‐induced dorsalization occurred only in cells injected with Wnt8 mRNA. These experiments suggest that the Wnt8 protein acts within the cell, likely in the ER or on the cell surface in an autocrine manner for dorsalization.     

Wnt/β‐catenin‐signaling and the formation of Müller glia‐derived progenitors in the chick retina

Müller glia can be stimulated to de‐differentiate, proliferate, and form Müller glia‐derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de‐differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt‐pathway. The purpose of this study was to investigate how Wnt‐signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA‐damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt‐signaling, including dkk1, dkk3, axin2, c‐myc, tcf‐1, and cd44. We find accumulations of nuclear β‐catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA‐treatment. Inhibition of Wnt‐signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt‐signaling, with GSK3β‐inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt‐signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK‐signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β‐catenin. In FGF2‐treated retinas, inhibition of Wnt‐signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt‐signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt‐signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt‐signaling is required for the formation of proliferating MGPCs. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 983–1002, 2016     

Requirement for Wnt and FGF signaling in Xenopus tadpole tail regeneration

We have investigated the requirement for the FGF and Wnt/beta-catenin pathways for Xenopus tadpole tail regeneration. Pathways were modified either by treatment with small molecules or by induction of transgene expression with heat shocks. Regeneration is inhibited by treatment with the FGF inhibitor SU5402, or by activation of a dominant negative FGF receptor, or by activation of expression of the Wnt inhibitor Dkk1. Agents promoting Wnt activity: the small molecule BIO, or a constitutively active form of beta-catenin, led to an increased growth rate. Combination of a Wnt activator with FGF inhibitor suppressed regeneration, while combination of a Wnt inhibitor with a FGF activator allowed regeneration. This suggests that the Wnt activity lies upstream of the FGF activity.Expression of both Wnt and FGF components was inhibited by activation of noggin, suggesting that BMP signalling lies upstream of both Wnt and FGF.The results show that the molecular mechanism of Xenopus tadpole tail regeneration is surprisingly similar to that of the Xenopus limb bud and the zebrafish caudal fin, despite the difference of anatomy.     

Neural induction requires BMP inhibition only as a late step, and involves signals other than FGF and Wnt antagonists

A dominant molecular explanation for neural induction is the 'default model', which proposes that the ectoderm is pre-programmed towards a neural fate, but is normally inhibited by endogenous BMPs. Although there is strong evidence favouring this in Xenopus, data from other organisms suggest more complexity, including an involvement of FGF and modulation of Wnt. However, it is generally believed that these additional signals also act by inhibiting BMPs. We have investigated whether BMP inhibition is necessary and/or sufficient for neural induction. In the chick, misexpression of BMP4 in the prospective neural plate inhibits the expression of definitive neural markers (Sox2 and late Sox3), but does not affect the early expression of Sox3, suggesting that BMP inhibition is required only as a late step during neural induction. Inhibition of BMP signalling by the potent antagonist Smad6, either alone or together with a dominant-negative BMP receptor, Chordin and/or Noggin in competent epiblast is not sufficient to induce expression of Sox2 directly, even in combination with FGF2, FGF3, FGF4 or FGF8 and/or antagonists of Wnt signalling. These results strongly suggest that BMP inhibition is not sufficient for neural induction in the chick embryo. To test this in Xenopus, Smad6 mRNA was injected into the A4 blastomere (which reliably contributes to epidermis but not to neural plate or its border) at the 32-cell stage: expression of neural markers (Sox3 and NCAM) is not induced. We propose that neural induction involves additional signalling events that remain to be identified.     

Increased activity of mesenchymal ALK2‐BMP signaling causes posteriorly truncated microglossia and disorganization of lingual tissues

Proper development of taste organs including the tongue and taste papillae requires interactions with the underlying mesenchyme through multiple molecular signaling pathways. The effects of bone morphogenetic proteins (BMPs) and antagonists are profound, however, the tissue‐specific roles of distinct receptors are largely unknown. Here, we report that constitutive activation (ca) of ALK2‐BMP signaling in the tongue mesenchyme (marked by Wnt1‐Cre) caused microglossia—a dramatically smaller and misshapen tongue with a progressively severe reduction in size along the anteroposterior axis and absence of a pharyngeal region. At E10.5, the tongue primordia (branchial arches 1–4) formed in Wnt1‐Cre/caAlk2 mutants while each branchial arch responded to elevated BMP signaling distinctly in gene expression of BMP targets (Id1, Snai1, Snai2, and Runx2), proliferation (Cyclin‐D1) and apoptosis (p53). Moreover, elevated ALK2‐BMP signaling in the mesenchyme resulted in apparent defects of lingual epithelium, muscles, and nerves. In Wnt1‐Cre/caAlk2 mutants, a circumvallate papilla was missing and further development of formed fungiform papillae was arrested in late embryos. Our data collectively demonstrate that ALK2‐BMP signaling in the mesenchyme plays essential roles in orchestrating various tissues for proper development of the tongue and its appendages in a region‐specific manner.     

BMP antagonists and FGF signaling contribute to different domains of the neural plate in Xenopus

In ectodermal explants from Xenopus embryos, inhibition of BMP signaling is sufficient for neural induction, leading to the idea that neural fate is the default state in the ectoderm. Many of these experiments assayed the action of BMP antagonists on animal caps, which are relatively naïve explants of prospective ectoderm, and different results have led to debate regarding both the mechanism of neural induction and the appropriateness of animal caps as an assay system. Here we address whether BMP antagonists are only able to induce neural fates in pre-patterned explants, and the extent to which neural induction requires FGF signaling. We suggest that some discrepancies in conclusion depend on the interpretations of sox gene expression, which we show not only marks definitive neural tissue, but also tissue that is not yet committed to neural fates. Part of the early sox2 domain requires FGF signaling, but in the absence of organizer signaling, this domain reverts to epidermal fates. We also reinforce the evidence that ectodermal explants are naïve, and that explants that lack any dorsal prepattern are readily neuralized by BMP antagonists, even when FGF signaling is inhibited.     

Neural induction: New achievements and prospects

Neural induction is a triggering of neural differentiation in a portion of cells of the vertebrate embryonic ectoderm in response to signals emanating from adjacent tissues. As revealed more than ten years ago in experiments with Xenopus embryos, the major role in neural induction is played by suppression of the bone morphogenetic protein (BMP) signaling cascade in neural cell precursors. Consequently, the epidermal differentiation program is blocked and a neural program is activated in such cells by default. The so-called default model of neural induction was supported with other experimental subjects. An important role in neural induction is also played by the FGF and Wnt signaling cascades via their interactions with the BMP cascade. As new regulatory proteins involved in neural induction were identified and their properties analyzed in detail, it became possible to apply mathematical modeling to study, with the example of neural induction, the spatial self-organization of cell differentiation in the embryo as one of the main problems of developmental biology.     

Active signals,gradient formation and regional specificity in neural induction

The question of how the vertebrate embryo gives rise to a nervous system is of paramount interest in developmental biology. Neural induction constitutes the earliest step in this process and is tightly connected with development of the embryonic body axes. In the Xenopus embryo, perpendicular gradients of BMP and Wnt signals pattern the dorsoventral and anteroposterior body axes. Both pathways need to be inhibited to allow anterior neural induction to occur. FGF8 and IGF are active neural inducers that together with BMP and Wnt signals are integrated at the level of Smad 1/5/8 phosphorylation. Hedgehog (Hh) also contributes to anterior neural induction. Suppressor-of-fused plays an important role in intertwining the Hh and Wnt pathways. Distinct mechanisms are discussed that establish morphogen gradients and integrate retinoic acid and FGF signals during posterior development. These findings not only improve our understanding of regional specification in neural induction, but have profound implications for mammalian stem cell research and regenerative medicine.     

PAWS1 controls Wnt signalling through association with casein kinase 1α

The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co‐localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

FGFs, Wnts and BMPs mediate induction of VEGFR-2 (Quek-1) expression during avian somite development

Regulation of VEGFR-2 (Quek1) is an important mechanism during blood vessel formation. In the paraxial mesoderm, Quek1 expression is restricted to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube. By implanting FGF 8b/8c or SU 5402 beads into the paraxial mesoderm, we show that FGF8 in addition to BMP4 from the intermediate mesoderm (IM) is a positive regulator of VEGFR-2 (Quek1) expression in the quail embryo. The expression of Quek1 in the medial somite half is normally repressed by the notochord and Sfrps-expression in the neural tube. Over-expression of Wnt 1/3a also results in an up-regulation of Quek1 expression in the somites. We also show that up-regulation of FGF8/Wnt 1/3a leads to an increase in the number of endothelial cells, whereas inhibition of FGF and Wnt signaling by SU 5402 and Sfrp-2 results in a loss of endothelial cells. Our results demonstrate that the regulation of Quek1 expression in the somites is mediated by the cooperative actions of BMP4, FGF8 and Wnt-signaling pathways.